BG108228A - RECOMBINANT ANTIGENES RELATED TO GANGLIOSIDE AND THEIR APPLICATION IN DIAGNOSTICING AND TREATMENT OF TUMORS - Google Patents

Abstract

The invention relates to the obtention of genetically-engineered antibodies from the P3 (AcM P3) murine monoclonal antibody, which is produced by the hybridoma deposited under number ECACC 94113026 inn accordance with the Budapest Treaty, and from the 1E10 (AcMai 1E10)) anti-idiotype thereof, which is produced by hybridoma ECACC 97112901 with the purpose of obtaining antibodies which have the same recognition properties as the original but which are less immunogenic. The chimaeric antibodies contain the variable domains of murine immunoglobulin and the constant regions of human immunoglobulin. The humanised antibodies, in addition to containing the constant regions of human immunoglobulin, are modified in the murine framework (FRs) region, and, in particular, in those areas that can be converted into a T-cell antigenic site, as a result of which some FRs positions are human. Said antibodies can be used in the diagnosis and therapy of different types of tumours.

Description

Technical field

The present invention relates to the biotechnological field, in particular to novel recombinant antibodies obtained by genetic engineering, in particular to chimeric and humanized antibodies derived from mouse P3 monoclonal antibody (MAb P3) and its anti-idiotypic monoclonal antibody10 (MAbai 1E10).

In particular, the present invention relates to antibodies that bind to gangliosides containing N-glycolated sialic acid, but does not refer to acetylated forms of gangliosides or to asexual glycolipids. Gangliosides containing N-glycolated sialic acid,

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are antigens widely manifested in breast cancer and melanomas. On the other hand, the antitumor effect of MAbai 1E10 in experimental models is also shown.

The present invention also relates to pharmaceutical compositions containing previously described recombinant antibodies useful in the diagnosis and treatment of cancer, in particular breast and melanoma cancers.

BACKGROUND OF THE INVENTION

Gangliosides are glycosphingolipids that contain sialic acid and are present in the plasma membrane in vertebrate cells (Stulls et al. (1989): Glycosphingolipids: structure, biological source and properties, Methods Enzymology, 179: 167-214). Some of these molecules have been described in the literature as antigens relating to tumors or tumor markers (Hakomory et al. (1991): Possible functions of tumor associated carbohydrate antigens, Curr. Opin. Immunol., 3: 646-653), as therefore, the administration of anti-ganglioside antibodies has been described as effective in the diagnosis and treatment of cancer (Hougton et al. (1985): Mouse monoclonal antibody lgG3 antibody detecting GD3 ganglioside: to phase I trial in patients with malignant melanoma, PNAS USA , 82: 1242-1246; Zhang et al. (1997): Selection of carbohydrate tumor antigens as targets for immune attack using immunohistochemistry. I. Focus on gangliosides, I nt. J. Cancer, 73: 42-49).

Sialic acids, more commonly expressed in animals, are acetyl (NeuAc) and N-glycolyl (NeuGc) (Gorfield et al. (1982): Occurrence of sialic acids, Cell. Biol. Monogr., 10: 5-50). N-glycolyl (NeuGc) is not generally expressed in normal human and chicken tissues, but is widespread in other vertebrates (Leeden and Yu, (1976): Chemistry and analysis of sialic acid. In: Biological Role of Sialic Acid. Rosemberg A and Shengtrund CL (Eds), Plenum Press, New York, 1-48; Kawai et al. (1991):

Quantitative determination of N-glycolylneuraminic acid expression in human cancerous tissues and avian lymphoma cell lines as tumor-associated sialic acid by gas chromatography-mass spectrometry, Cancer Research, 51: 1242-1246). However, there are articles showing that anti-NeuGc antibodies recognize some tumors and cell lines in humans (Higashi et al. (1988): Detection of gangliosides as N-glycolylneuraminic acid specific tumor-associated Hanganutziu-Deicher antigen in human retinoblastoma cells, Jpn. J. Cancer Res., 79: 952-956; Fukui et al. (1989): Detection of glycoproteins as a tumor-associated Hanganutziu-Deicher antigen in human gastric cancer cell line, NUGC4, Biochem. ., 160: 1149-1154). Elevated levels of GM3 (NeuGc) gangliosides have been found in human breast cancer (Marquina et al. (1996): Gangliosides expressed in human breast cancer, Cancer Research, 1996; 56: 51655171), which makes this application attractive. molecule as a target for cancer treatment.

The monoclonal antibody (MAb) P3 produced by the cell line with accession number ECACCS 94113026 (European Patent EP 0 657 471 B1) is a mouse monoclonal antibody of the IgM isotype, which is obtained by fusion of murine splenocytes from BALB / c mice, immunized with liposomes containing GM3 (NeuGc) and tetanus toxoid, with cell line P3-X63Ad8.653; which is myeloma in a mouse. This monoclonal antibody MAb P3 strongly interacts with gangliosides containing N-glycolated sialic acid, but does not interact with the acetylated forms of gangliosides or with asexual glycolipids. It has been shown by immunocytochemical and immunohistochemical studies conducted with cell lines and tissues from benign and neoplastic tumors that the monoclonal antibody MAb P3 recognizes breast cancer (Vazquez et al. (1995): Generation of a murine monoclonal acid specific for N -containing gangliosides that also recognizes sulfated glycolipids (Hybridoma, 14: 551-556) and melanoma.

Mab P3 monoclonal antibody induces anti-idiotypic immune response (Aβ2) in BALB / c mice (syngenetic model), even without accessory and carrier protein (Vazquez et al. (1998): Syngeneic anti-idiotypic monoclonal antibodies to an anti-NeuGside-containing antibodies monoclonal antibody, Hybridoma, 17: 527-534). The role of electronegative groups, sialic acid (for gangliosides) and SO ₃ '(for sulfatides) in recognizing the properties of this antibody, was demonstrated by immunochemical analysis (Moreno et al. (1998): Delineation of an epitope recognized by an antibody specific for N -glycolylneuraminic acid-containing gangliosides, Glycobiology, 8: 695-705).

The anti-idiotypic Mab 1E10 mouse monoclonal antibody (Mabai 1E10) of lgG1 subtype was obtained from BALB / c mouse immunized with KLH-associated Mab P3 monoclonal antibody (US Patent 6,063,379, cell line deposited under accession number ECACC 9711201). The Mabai 1E10 monoclonal antibody specifically recognizes MAb P3 and does not bind other IgM antiganglioside antibodies. Moreover, Mabai 1E10 monoclonal antibody inhibits the specific binding of Mab P3 monoclonal antibody to GM3 (NeuGc) and the MDA-MB-435 cell line derived from ductal breast carcinoma (positive for Mab P3 monoclonal antibody binding). The MAbai 1E10 monoclonal antibody induces a strong immune response to Aβ3 antibodies when immunized with mice of a syngenic or allogeneic model, and these Aβ3 antibodies do not show the same specificity as the Mab P3 monoclonal antibody, even when carrying idiopathic antibodies similar to the1 antibodies. (Vazquez et al. (1998): Syngeneic anti-idiotypic monoclonal antibodies to an anti-NeuGc-containing ganglioside monoclonal antibody, Hybridoma, 17: 527-534). The MAbai 1E10 monoclonal antibody induces a strong antitumor effect in both syngenetic and allogeneic mice. Growth of the F3II breast carcinoma cell line was significantly inhibited by a repeated dose of MAbai 1E10 monoclonal antibody bound to KLH in Freund supplementation when BALB / c mice were vaccinated.

Following vaccination, the number of spontaneous pulmonary metastases also decreases. The intravenous administration of MAbai 1E10 monoclonal antibody to C57BL / 6 inoculated mice, 10 to 14 days after intravenous inoculation of melanoma B16 cells, causes a dramatic decrease in the number of pulmonary metastases compared to mice treated with lateral IgG. These results indicate that there is more to one mechanism of antitumor activity (Vazquez et al. (2000): Antitumor properties of an anti-idiotypic monoclonal antibody in relation to N-glycolyl-containing gangliosides, Oncol. Rep., 7: 751 -756, 2000).

Even when hybridoma technology has been developed for 15 years (Koehler & Milstein (1975): Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 256: 495-497) and when monoclonal antibodies are already widely used in insertion of diagnoses, as well as in the scientific field, they do not show therapeutic efficacy for humans. This is mainly due to their short half-life in the blood and to the fact that the action functions in mice are not fulfilled in the human immune system, also in the human immune response to the anti-mouse antibody (NAMA reaction).

Otherwise, genetic engineering technology has discovered the power of using the monoclonal antibody MAb, since the production of modified antibodies with reduced antigenicity may be obtained by manipulating immunoglobulin genes, as well as improving the function of action in the treatment or diagnosis of certain pathologies. Immunoglobulin immunogenicity reduction methods have the primary objective of reducing the difference between mouse antibody and human immunoglobulin without altering the antigenic specificity of recognition (Morrison u Oi (1989): Genetically engineered antibody molecules, Adv. Immunol., 44: 65-92 ).

Recently, a number of methods have been developed for humanizing antibodies from mice or rats, and thus reducing the xenogenic immune response to foreign proteins when injected into humans. One of the first ways of reducing antigenicity was the chimeric antibodies, in which, in constant domains of human molecules, different domains of the murine protein were introduced, showing the same specificity but decreased immunogenicity compared to their murine counterparts as the human functions of action is stored by chimeric antibodies (Morrison et al. (1984): Chimeric human antibody molecules: Mouse antigen-binding domains with human constant region domains, PNAS USA, 81: 6851-6855). Even when the chimeric antibodies have the same specificity as the murine counterpart, an immune response to the variable regions of the rodent is often observed.

In an attempt to further reduce the immunogenicity of chimeric antibodies, only a CDR from a rodent monoclonal antibody can be grafted on a human basis and this variable hybrid region manifests itself in human constant regions (Jones et al. (1986): Replacing the complementarydetermining regions in a human antibody with those of a mouse, Nature, 321: 522524; Verhoeyen et al. (1988): Reshaping human antibodies: grafting an antilysozyme activity, Science, 239, 1534-1536). However, this method does have some disadvantages: the frequently obtained antibody lowers affinity and a number of frame residues need to be reversed to the corresponding murine residues to restore binding (Rietchmann et al. (1988): Reshaping human antibodies lor therapy, Nature, 332: 323-327; Queen et al. (1989): A humanized antibody that binds to the interleukin 2 receptor, PNAS USA 86: 10029-10033; Tempest et al. (1991): Reshaping a human monoclonal antibody to inhibit human respiratory syncytial virus infection in vivo, Biotechnology, 9: 266-272). Moreover, persistent immunogenicity is often observed in antibodies with CDR grafts.

Mateo et al. (U.S. Patent No. 5,712,120) describe a method of reducing the immunogenicity of murine antibodies. According to the method, the modification is limited to variable domains and, in particular, to murine frames of chimeric antibodies. Moreover, substitutions are made only in those areas of the amphipathic framework

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sequences and, hence, are potential epitopes recognized by T cells. The method involves the reasonable replacement of some amino acid residues localized in potential immunogenic epitopes by the corresponding residues of the most homologous human sequence, and the amino acids that are mainly responsible for the accepted structures and for residues adjacent to the CDR or in the Vernier region may be detained.

The resulting antibody retains its antigen binding specificity and is less immunogenic than its mouse or chimeric precursor (Mateo et al. (2000): Removal of T cell epitopes from genetically engineered antibodies: Production of modified immunoglobulins with reduced immunogenicity, Hybridoma, 19: 463-471) as these properties enhance their therapeutic application. Using this new method, only a few mutations and, of course, fewer genetic manipulations have to be performed.

SUMMARY OF THE INVENTION

The present invention relates to recombinant antibodies obtained by genetic engineering. In particular, the present invention relates to a chimeric antibody derived from mouse P3 monoclonal antibody produced by a hybridoma cell line with accession number ECACC 94113026. MAB P3 recognizes an antigen expressed in breast tumor cells and melanomas. The MAb P3 mouse monoclonal antibody is characterized by the following heavy and light chain hypervariable region (CDR) sequences:

HEAVY CHAIN

CDR1: RYSVH

CDR2: MIWGGGSTDYNSALKS

CDR3: SGVREGRAQAWFAY

CADENA LIGERA

CDR1: KASQDVSTAVA

CDR2: SASYRYT

CDR3: QQHYSTPWT

It is preferred that the framework (FR) sequences of the heavy and light chains are as follows:

HEAVY CHAIN

FR1: QVQLKESGPGLVAPSQSLSITCTVSGFSLS

FR2: WVRQPPGKGLEWLG

FR3: RLSISKDNSKSQVFLKMNSLQTDDTAMYYCAR

FR4: WGQGTLV

LIGHT CHAIN

FR1: DIVMTQSHKFMSTSVGDRVSITC

FR2: WYQQKPGQSPKLLIY

FR3: GVPDRRGSGSGTDRRISSVQAEDLAVYYC

FR4: FGGGTKL

In a preferred aspect, the chimeric antibody of the present invention contains a constant region in the heavy chain of human IgG1 and a constant region in the light chain of human Ck. In another aspect, the present invention relates to a humanized antibody derived from the Mab P3 monoclonal antibody produced by a hybridoma cell line with entry ECACC 94113026, characterized in that it contains a constant region in the human IgG1 heavy chain and a constant region in the human CK light chain and the light chain framework (FR) regions contain some of the following mutations:

LIGHT CHAIN

Item 8: His replaced by Pro

Item 9: Lys replaced by Ser

Item 10: Phe replaced by Ser

Item 11: Met replaced by Leu

Item 13: Thr replaced by Ala

In another aspect, the present invention relates to a chimeric antibody derived from mouse monoclonal antibody 1E10, produced from a hybridoma cell line with accession number ECACC 97112901, and is an anti-idiotypic antibody that recognizes the monoclonal antibody of MAb P3 mouse. The MAbai 1E10 anti-idiotypic mouse monoclonal antibody is characterized by the following heavy and light chain hypervariable region (CDR) sequences:

© HEAVY CHAIN

CDR1: SYDIN

CDR2: WIFPGDGSTKYNEKFKG

CDR3: EDYYDNSYYFDY

LIGHT CHAIN

CDR1: RASQDISNYLN

CDR2: YTSRLHSG

CDR3: QQGNTLPWT

It is preferred that the heavy and light & heavy chain (FR) sequences are as follows:

HEAVY CHAIN

FR1: QVQLQQSGAELVKPGASVKLSCKASGYTFT

FR2: WVRQRPEQGLEWIG

FR3: KATLTTDKSSSTAYMQLSRLTSEDSAVYFCAR

FR4: WGQGTTLTV

LIGHT CHAIN

FR1: DIQMTQTTSSLSASLGDRVTISC

FR2: WYQQKPDGTVKLLIY

FR3; VPSRFSGSGSGTDYSLTISNLEQEDIATYFC

FR4: FGGGTKLESK

In a preferred aspect, the chimeric antibody of the present invention contains a constant region in the heavy chain of human IgG1 and a constant region in the light chain of human Ck. In another aspect, the present invention relates to a humanized antibody derived from the Mab 1E10 mouse monoclonal antibody produced by a hybridoma cell line with entry ECACC 97112901, characterized in that it contains a constant region in the human IgG1 heavy chain and a constant region in the human CK light chain and the heavy and light chain framework (FR) regions contain some of the following mutations:

© LIGHT CHAIN

Item 7: Thr replaced by Ser

Item 8: Thr replaced by Pro

Item 15: Leu replaced by Val

HEAVY CHAIN

Item 5: Gin replaced by Val

Item 40: Arg replaced by Ala

Item 42: Glu replaced by Gly

Position 87 (83 according to Kabat numbering): Thr replaced by Arg * In another aspect, the present invention relates to cell lines expressing the chimeric and humanized antibodies described; the present invention also relates to pharmaceutical compositions containing the antibodies described.

Preferably, the present invention relates to pharmaceutical compositions for the treatment of breast cancer, lung cancer, digestive tract cancer, urogenital cancer, melanomas, sarcomas and neuroectodermal tumors, their metastases and recurrence, characterized in that they include those described antibodies and a suitable excipient.

In another aspect of the present invention, the pharmaceutical compositions can be used to locate and in diagnose breast cancer, lung cancer, digestive tract cancer, urogenital cancer, melanomas, sarcomas and neuroectodermal tumors, their metastases and recurrence, characterizing them by including the antibodies described.

CDNA synthesis and gene expansion by polymerase chain reaction (PCR) of the mouse MAb P3 variable region monoclonal antibody to its anti-idiotype monoclonal antibody MAbai 1E10

Cytoplasmic ribonucleic acid was extracted from about 10 ⁶ hybridoma cells of P3 (mouse IgM Mab that recognizes GM3 N-glycolated ganglioside) or 1E10 (anti-idiotypic anti-P3 antibody). Ribonucleic acid was extracted with TRIZOL reagent (GIBCO BRL, Grand Island, NY) according to the manufacturer's instructions.

The cDNA synthesis reaction was performed by mixing 5 pg of ribonucleic acid, 25 pmol Vh (addition to the murine IgM constant region for VHP3 and murine lgG1 constant region for VH 1E10) or Vk (addition to the constant murine kappa region for both antibodies), 2.5 mM of each dNTP, 50 mM Tris-HCI pH 7.5, 75 mM potassium chloride, 10 mM DTT, 8 mM magnesium dichloride and 15 units of ribonucleic acid inhibitor in 50 μΙ reaction mixture. The mixture was heated at 70 ° C for 10 minutes and slowly cooled to 37 ° C. Then, 100 units of MLV reversible transcriptase enzyme were added and incubation at 42 ° C continued for 1 hour.

The cDNA variable regions VK and VH are expanded upon polymerase chain reaction (PCR). Briefly, 5 μΙ cADN of VH or VK was mixed with 25 pmol of specific precursors, 2.5 mM of each dNTP, 5 μΙ of the ingredients of 10X Taq DNA polymerase buffer, and 1 unit of this enzyme. Samples were subjected to temperature cycles at 94 ° C for 30 seconds; 50 ° C, 30 seconds; 72 ° C, 1 minute; and last incubation for 5 minutes at 72 ° C.

Cloning and sequence of the expanded cDNA

The polymerase chain reaction (PCR) products VH and VK (respectively P3 and 1E10) were cloned into a TA vector (TA Cloning kit. Promega, USA). The resulting clones were sequenced by dideoxy method using T7 DNA polymerase (T7 Sequencing kit. Pharmacia, Sweden).

φ ^w Construction of chimeon genes

VH and VK genes are taken from TA vectors by enzymatic digestion and cloned into corresponding expression vectors (Coloma et al. (1992): Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction, J. Immunol. Meth., 152: 89-104).

VH genes are harvested from the TA vector by enzymatic digestion with EcoRV and Nhel and cloned into an expression vector (PAS 4604) that includes a variable region of human IgG1 and a histidinol-resistant gene. © The resulting structures are P3VH-PAH4604 and 1E10VH-PAH4604. VK genes are taken from the TA vector by enzymatic digestion with EcoRV and Sall and cloned into an expression vector (PAG 4622). This vector includes the mycophenolic acid-resistant gene and the human kappa constant region. The resulting constructs are P3VK-PAG4622 and 1E10VK-PAG4622.

Expression of chimeric antibodies derived from mouse monoclonal antibody (MAb P3) and Mabid 1E10

NS-0 cells were electroporated with 10 pg P3VK-PAG4622 or 1E10VKPAG4622, and clones expressing human cap light chains were transferred to 10 pg P3VH-PAH4604 or 1E10VH-PAH4604.

The deoxyribonucleic acids are linearized by digestion with the Pvul enzyme, precipitated with ethanol and dissolved in 50 μΙ PBS. Approximately 10 ⁷ cells were seeded by centrifugation and resuspended in 0.5 ml PBS together with the digested DNA in the electroporation cuvette. After 10 minutes on ice, a pulse of 200 volts and 960 pF was placed on the cells and left in ice for another 10 minutes. Cells were distributed in a 96-well plate with D'MEM F12 plus 10% fetal calf serum. After two or four days, a selective medium (D'MEM F12 with mycophenolic acid 0.45 pg / ml or histidinol mM, respectively) was added. The clones become visible to the naked eye after 14 days.

The presence of a human antibody in the middle of the wells containing the transferred clones is measured by ELISA. The microtiter plate wells are coated with goat anti-human kappa light chains (for human kappa chain producing clones) or anti-human IgG (specific gamma chain). of antibody clones) antibodies. After washing with PBST (buffered saline phosphate solution containing 0.05% Tween 20), diluted culture media from the transfectant containing wells was added to each microtiter well for 1 hour at 37 ° C. The wells were washed with PBS-T and peroxidase was added from horseradish-conjugated goat anti-human kappa light chain or alkaline phosphatase-bound goat anti-human IgG (specific gamma chain) and then incubated for 1 hour at 37 ° C. The wells were washed with PBS-T and substrate buffer containing o-phenylenediamine or rnitrophenyl phosphate was added, respectively. After half an hour, an absorption of 492 or 405 pt was measured, respectively.

Construction of humanized P3hu and 1 E10hu antibodies in the humanization of T cell epitopes. Prediction of T cell epitopes

The sequences of the variable domains of P3 and 1E10 were analyzed using the AMPHI algorithm (Margalit et al. (1987): Prediction of immunodominant helper T cell antigenic sites from the primary sequence, J. Immunol., 138: 2213-2229). He is looking for spiral amphipathic segments with 7 or 11 amino acid residues that bind to T immunogenicity. The SOHHA program also envisages spiral hydrophobic segments. (Elliot et al. (1987): An hypothesis on the binding of an amphipatic, alpha helical sequence in the class II antigen desotope, J. Immunol., 138: 2949-2952). Both algorithms predict which segments of the variable region of antibodies P3 and 1E10 can represent the β-helper cells in the context of MHC class II molecules.

Homologous analysis with human immunoglobulins

The amino acid sequences of the murine variable regions are compared with the immunoglobulin sequences included in the GeneBank and EMBL database (available online). The homologous sequence of the human variable region is determined for each antibody. Software PC-TWO HIBIO PROSIS 06-00 (Hitachi) was used to search for the homologous sequence.

Immunogenicity reduction assay

The purpose of the method is to reduce immunogenicity, destroying or humanizing potential immunogenic T epitopes, with minimal changes. The method involves the reasonable replacement of some amino acid residues localized in helical amphipathic segments. The amino acids that are mainly responsible for the accepted structures as well as residues in the immediate vicinity of the CDR or in the Vernier zone must be retained.

According to this method, the mouse variable region sequences are compared with the most homologous human sequence, identifying different amino acid residues at each position between mouse MAb and the most homologous human sequence, with only residues within being considered (Kabat (1991) Immunological Interest Sequences (Fifth Edition, National Institute of Health), replacing previously defined residues with those residues present in the most homologous human sequence. Changes are made by direct mutagenesis methods.

The residues included in the three-dimensional structure of the junction site do not change; they may affect antigen recognition. Further information on the effect of substitutions in the structure can be obtained by molecular modeling of the antigen binding site.

The presence of proline residues in the helical amphipathic segment should be considered, and the fact that some murine residues do not occur at the same position in the most homologous human sequence but are common in other human immunoglobulins. Therefore, there is no consensus on the substitution of murine amino acids within. Different versions of modified antibodies may be obtained with different numbers of substitutions. Mutations are performed by PCR overlap.

Cloning and expression of humanized antibodies P3hu and 1E10hu

Genetic constructs corresponding to P3hu and 1E10hu are cloned into expression vectors by the method described above for chimeric antibodies. The constructs obtained are P3VKhu-PAG4622 or 1E10VKhuPAG4622 and P3VHhu-PAH4604 or 1E10VHhu-PAH4604. They are transferred to NS-0 cells following the protocol described above for chimeric antibodies.

Purification of recombinant antibodies

Recombinant antibodies were purified by affinity chromatography using protein A (Pharmacia, Upssala, Sweden).

Biological activity

Specific antigen binding was measured by ELISA, in which the biological activity of the recombinant antibodies was tested.

For the recombinant MAb P3 antibody, the microtiter plates were coated with GM3 (NeuGc) ganglioside in methanol. After drying for one hour, nonspecific binding was blocked with bovine sera albumin (BSA) 1% in Tris-HCl buffer and incubated for 1 hour at 37 ° C. The wells were washed with PBS and incubated for 1 hour at 37 ° C with purified recombinant MAb P3. The wells were washed with Tris-HCl, goat anti-human antibody bound to alkaline phosphatase was added and incubated for 1 hour at 37 ° C. Finally, the wells were washed and substrate buffer containing p-nitrophenyl phosphate was added. After half an hour, the absorbance was measured at 405 or 492 nm, respectively.

For the recombinant Mabai 1E10 antibody, the EUSA analysis is the same, except that the wells are coated with MAb P3 and washing is performed with PBS-0.05% Tween 20.

Examples of carrying out the invention

In the following examples, all enzymes used, as well as reagents and materials, are commercial products, unless otherwise noted.

Example 1. Preparation of Chimeric MAb P3

The synthesis of cDNA is carried out by reacting with the reversible transcriptase enzyme, starting from ribonucleic acid from a hybridoma producing MAb P3, as described above. The sequences of the specific precursors used in this reaction are as follows:

For VH:

5AGGTCTAGAA (CT) CTCCACACACAGG (AG) (AG) CCAGTGGATAGAC 3 '

For VK:

5′GCGTCTAGAACTGGATGGTGGGAAGATGG 3 ′ cDNA VHP3 and cDNA VKP3 are amplified by PCR using Taq Polymerase and specific precursors. The restriction sites included in the precursors are ECORV / NHEI for VH and ECORV / SALI for VK. The precursor sequences used are as follows:

For VH:

Precursor 1 (signal peptide):

5'GGGGATATCCACCATGG (AG) ATG (CG) AGCTG (TG) GT (CA) AT (CG) CTCTT3 'Precursor 2 (CH1):

5'GGGGCTAGCTGCAGAGACAGTGACCAGAGT3 '

For VK:

Precursor 1 (signal peptide):

5'GGGGATATCCACCATGGAG (TA) CACA (GT) (TA) CTCAGGTCTTT (GA) T 3 'Precursor 2 (Sk):

5'AGCGTCGACTTACGTTT (TG) ATTTCCA (GA) CTT (GT) GTCCC 3 '

PCR products were cloned into TA vector (TA cloning kit, Invitrogen). Twelve independent clones were sequenced by dideoxy method using T7 DNA Pol (Pharmacia). Homologous search analysis determines the most homologous group in the sequence for VHP3 and VKP3. The VHP3 and VKP3 sequences (Figures 1 and 2) have high homology, respectively with the groups IB and V, according to the Kabat classification.

After digestion with the restricted ECORV and NHEI enzymes for VHP3 and with ECORV and SALI for VKP3, they are cloned into expression vectors previously digested with the same enzymes, PAN4604 and PAG4622, respectively for VH and VK. These expression vectors were donated by Sherie Morrison (UCLA, California, USA) and are suitable for expression of immunoglobulins in mammalian cells. Vector PAS4604 includes the human constant region lgG1 and PAG4622 (Coloma et al. (1992): Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction, J. Immunol. Meth. 152: 89-104). The resulting constructs are P3VH-PAH4604 and P3VKPAG4622.

NS-0 cells were infected with 10 µg P3VK-PAG4622, a light chain expressing clone was infected with 10 µg P3VH-PAH4604, and in both cases deoxyribonucleic acid was linearized with Pvul, precipitated with ethanol and dissolved in 50 µL PBS before transfection.

Approximately 10 ⁷ cells were seeded by centrifugation and resuspended in 0.5 ml PBS together with the digested deoxyribonucleic acid in the electroporation cuvette. After 10 minutes on ice, a pulse of 200 volts and 960 cP was placed on the cells and left in ice for another 10 minutes. Cells were distributed in a 96-well plate with D'MEM F12 plus 10% fetal calf serum. After two or four days a selective medium (D'MEM F12 with mycophenolic acid 0.45 µg / ml or histidinol 10 mM, respectively) was added. Transfected clones become visible to the naked eye after 14 days.

The presence of a human antibody in the middle of the wells containing the transferred clones is measured by ELISA. The wells of the microtiter plate are coated with goat anti-human kappa light chains (for human kappa chain producing clones) or anti-human IgG (specific gamma chain). of antibody clones) antibodies. After flushing with

PBST (saline phosphate buffered solution containing 0.05% Tween 20) was added to each microtiter well for 1 hour at 37 ° C and diluted culture media from the wells containing transfectants was added. The wells were washed with PBS-T and peroxidase was added from horseradish-conjugated goat anti-human kappa light chain or alkaline phosphatase-bound goat anti-human IgG (specific gamma chain) and then incubated for 1 hour at 37 ° C. The wells were washed with PBS-T and substrate buffer containing o-phenylenediamine or rnitrophenyl phosphate was added, respectively. After half an hour, an absorbance of 492 or 405 nm was measured, respectively.

Example 2, Preparation of different versions of humanized antibody P3

Mouse VHP3 is VKP3 sequences (Figures 1 and 2) are compared to human sequences. Figures 3 and 4 show the most homologous human sequences. Spiral amphipathic regions or potential epitopes of T cells are searched on murine sequences with a P3 variable region and, according to the method, a reasonable replacement of amino acids is performed to destroy or humanize potential epitopes of T cells to murine sequences.

Analysis of VHP3 yields (Figure 3) two amphipathic segments, the first segment covering CDR1, FR2 and some residues of CDR2, and the second segment covering the end of FR3 and CDR3. The major differences in the mouse sequence compared to the most homologous human sequence are found in the CDR or residues involved in the three-dimensional structure of the binding site. To this end, it was decided not to replace any amino acid in murine VHP3.

Analysis of VKP3 also yielded (Figure 4) two amphipathic segments, the first segment covering FR1 and the second segment covering CDR2 and some FR3 residues. It was decided to replace residues in positions 8, 9, 10, 11 and residues in the same position in the most homologous human sequence. The amino acids His, Lys, Phe, Met and Thr were replaced by Pro, Ser, Ser, Leu and Ala, respectively. Replacements were performed by PCR overlap (Kammann et al. (1989): Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR), Nucleic Acids Res., 17: 5404)) using precursors 1,2,3 and 4, the sequences of which are as follows:

Precursor 1:

5 'ATGACCCAGTCTCCTTCTTCTCTTTCCGCGTCAGTAGGAGAC 3'

Precursor 2:

5 'AGCGTCGACTTACGTn (TG) ATTTCCA (GA) CTT (GT) GTCCC 3'

Precursor 3:

5 'GTCTCCTACTGACGCGGAAAGAGAAGAAGGAGACTGGGTCAT 3'

Precursor 4:

5 'GGGGATATCCACCATGGAG (TA) CACA (GT) (TA) CTCAGGTCTTT (GA) T 3'

The mutation sites were confirmed when the sequences were created. The resulting construct is P3Vkhu and it is cloned into PAG 4622 expression vector. The resulting construction is P3Vkhu-PAG4622. For the development of humanized P3 antibody, NS-Cells were infected with P3VHPAP4604 and P3Vkhu-PAG4622.

The P3hu antibody was infected following the same method described above for chimeric antibodies.

Example 3. Biological Activity of Chimeric MAb P3

The specific binding to an antigen was measured by ELISA to determine the biological activity of chimeric MAb 3.

For the recombinant MAb P3 antibody, the microtiter plates were coated with GM3 (NeuGc) ganglioside in methanol. After drying for one hour, nonspecific binding was blocked with bovine sera albumin (BSA) 1% in Tris-HCl buffer and incubated for 1 hour at 37 ° C. The wells were washed with PBS and incubated for 1 hour at 37 ° C with purified recombinant Mab P3. The wells were washed with Tris-HCl, goat anti-human antibody bound to alkaline phosphatase was added and incubated for 1 hour at 37 ° C. Finally, the wells were washed and substrate buffer containing p-nitrophenyl phosphate was added. After half an hour, the absorbance at 405 nm was measured.

Chimeric Mab T1 is used for negative control.

Figure 5 shows the specific binding of chimeric MAb P3 to the antigen.

Example 4, Preparation of Chimeric MAb 1E10

The synthesis of cDNA is carried out by reacting with the reversible transcriptase enzyme, starting from ribonucleic acid from a hybridoma producing Mab 1E10, as described above. The sequences of the specific precursors used in this reaction are as follows:

For VH:

5'GGGGCTAGCTGAGGAGACTGTGAGAGTGGT 3 '

For VK:

5′GCGTCTAGAACTGGATGGTGGGAAGATGG 3 ′ cDNA VH1E10 and cDNA VK1E10 are amplified by PCR using Taq Polymerase and specific precursors.

For VH:

Precursor 1 (signal peptide):

5'GGGGATATCCACCATGG (AG) ATG (CG) AGCTG (TG) GT (CA) AT (CG) CTCTT3 'Precursor 2 (CH1):

5'GGGGCTAGCTGAGGAGACTGTGAGAGTGGT3 '

For VK:

Precursor 1 (signal peptide):

5'GGGGTTAACCACCATGAGG (GT) CCCC (AT) GCTCAG (CT) T (CT) CT (TG) GG (GA) 3 'Precursor 2 (Ck):

5'AGCGTCGACTTACGTTT (TG) ATTTCCA (GA) CTT (GT) GTCCC 3 '

PCR products were cloned into TA vector (TA cloning kit, Invitrogen). Twelve independent clones were sequenced (Figures 7 and 8) by dideoxy method using T7 DNA Pol (Pharmacia). The homologous search group determines the most homologous group in the sequence for VH1E10 and VK1E10. The VH1E10 and VK1E10 sequences have high homology, respectively with mixed groups and V, according to the Kabat classification.

After uptake by the restricted ECORV and NHEI enzymes for VH1E10 and Hindi and SALI for VK1E10, they are cloned into expression vectors previously digested with the same enzymes, PAN4604 and PAG4622, respectively for VH and VK. These expression vectors were donated by Sherie Morrison (UCLA California, USA) and are suitable for expression of immunoglobulins in mammalian cells. Vector PAS4604 includes the human constant region lgG1 and PAG4622 (Coloma et al. (1992): Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction, J. Immunol. Meth. 152: 89-104). The resulting structures are 1E10VH-PAH4604 and 1E10VK-PAG4622.

NS-0 cells were infected with 10 pg 1E10VK-PAG4622, a clone expressing light chain was infected with 10 pg 1E10VH-PAH4604, and in both cases deoxyribonucleic acid was linearized with Pvul, precipitated with ethanol and dissolved in 50 pl. PBS before transfection.

Approximately 10 ⁷ cells were seeded by centrifugation and resuspended in 0.5 ml PBS together with the digested deoxyribonucleic acid in the electroporation cuvette. After 10 minutes on ice, a pulse of 200 volts and 960 pF was placed on the cells and left in ice for another 10 minutes. Cells were distributed in a 96-well plate with D'MEM F12 plus 10% fetal calf serum. After two or four days, a selective medium (D'MEM F12 with mycophenolic acid 0.45 pg / ml or histidinol 10 mM, respectively) was added. Transfected clones become visible to the naked eye after 14 days.

The presence of a human antibody in the middle of the wells containing the transferred clones was measured by ELISA. The wells of the microtiter plate are coated with goat anti-human kappa light chains (for human kappa chain producing clones) or anti-human IgG (specific gamma chain) (for full production of antibody clones) antibodies. After washing with PBST (buffered saline phosphate solution containing 0.05% Tween 20), diluted culture media from the transfectant containing wells was added to each microtiter well for 1 hour at 37 ° C. The wells were washed with PBS-T and peroxidase was added from horseradish-conjugated goat anti-human kappa light chain or alkaline phosphatase-bound goat anti-human IgG (specific gamma chain) and then incubated for 1 hour at 37 ° C. The wells were washed with PBS-T and substrate buffer containing o-phenylenediamine or rnitrophenyl phosphate was added, respectively. After half an hour, an absorbance of 492 or 405 nm was measured, respectively.

Example 5. Preparation of different versions of humanized antibody P3

Mouse VH1E10 is VK1E10 sequences (Figures 6 and 7) compared to human sequences. Figures 8 and 9 show the most homologous human sequences. Spiral amphipathic regions or potential epitopes of T cells are searched for murine sequences with the 1E10 variable region and, according to the method, a reasonable replacement of amino acids is performed to destroy or humanize potential epitopes of T cells to murine sequences.

Analysis of VH1E10 yielded (Figure 8) three amphipathic segments, the first segment covering FR1, the second segment covering FR2, and the third segment covering FR3. It was decided to replace residues in positions 5, 40, 42 and 87 (83, according to the Kabat numbering) with residues in the same position in the most homogeneous human sequence. The amino acids Gin, Arg and Glu were replaced, respectively, by Val, Ala, Gly and Arg.

Replacements were performed by PCR overlap (Kammann et al. (1989): Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR), Nucleic Acids Res., 17: 5404) using a different set of precursors.

The heavy chain position mutation precursors are 1, 2, 3 and 4, the sequences of which are as follows:

Precursor 1:

5 'CAGGTTCAGCTGGTGCAGTCTGGAGCT 3'

Precursor 2:

5 'GGGGCTAGCTGAGGAGACTGTGAGAGTGGT 3'

Precursor 3:

5 'AGCTCCAGACTGCACCAGCTGAACCTG 3'

Precursor 4:

5 'GGGGATATCCACCATGG (AG) ATG (CG) AGCTG (TG) GT (CA) AT (CG) CTCTT3'

After checking the sequences at position 5, mutations at positions 40 and 42 are introduced.

Heavy chain position mutation precursor 40 and 42:

Precursor 1:

5 'TGGGTGAGGCAGGCGCCTGGGCAGGGACTTGAG 3'

Precursor 2:

5 'GGGGCTAGCTGAGGAGACTGTGAGAGTGGT 3'

Precursor 3:

5 'CTCAAGTCCCTGCCCAGGCGCCTGCCTCACCCA 3'

Precursor 4:

5 'GGGGATATCCACCATGG (AG) ATG (CG) AGCTG (TG) GT (CA) AT (CG) CTCTT3'

After checking the sequences at positions 40 and 42, a mutation at position 87 (83 according to Kabat numbering) was introduced.

Heavy-chain mutation precursor at position 87 (83, according to Kabat numbering):

Precursor 1:

5 'CTCAGCAGGCTGCGGTCTGAGGACTCT 3'

Precursor 2:

5 'GGGGCTAGCTGAGGAGACTGTGAGAGTGGT 3'

Precursor 3:

5 'AGAGTCCTCAGACCGCAGCCTGCTGAG 3'

Precursor 4:

5 'GGGGATATCCACCATGG (AG) ATG (CG) AGCTG (TG) GT (CA) AT (CG) CTCTT3'

Other substitutions were not made because the residues were introduced into the three-dimensional structure of the junction site.

The mutation sites were confirmed when the sequences were created. The resulting construct is 1E10VHhu and is cloned into PAS4604 expression vector. The resulting construction is 1E10VH-PAH4604.

Analysis of VK1E10 yielded (Figure 9) three amphipathic segments, the first segment covering FR1, the second segment covering CDR1, and the third segment covering FR3. It was decided to replace residues in positions 7, 8 and 15 with residues in the same position in the most homologous human sequence. The amino acids Thr, Thr and Leu were replaced, respectively, by Ser, Pro and Val. Replacements were performed by PCR overlap (Kammann et al. (1989): Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR), Nucleic Acids Res., 17: 5404) using precursors 1, 2, 3 and 4, whose sequences are as follows:

Mutation Chain Mutation Precursors 7, 8, and 15:

Precursor 1:

5'CAGATGACACAGTCTCCTTCCTCCCTGTCTGCCTCTGTGGGAGACAGAGTC 3 '

Precursor 2:

5'AGCGTCGACTTACGTTT (TG) ATTTCCA (GA) CTT (GT) GTCCC 3 '

Precursor 3:

5'GACTCTGTCTCCCACAGAGGCAGACAGGGAGGAAGGAGACTGTGTCATCTG 3 'Precursor 4:

5'GGGGTTAACCACCATGAGG (GT) CCCC (AT) GCTCAG (CT) T (CT) CT (TG) GG (GA) 3 '

The mutation sites were confirmed when the sequences were created. The resulting construct is 1E10Vkhu and it is cloned into a PAG4622 expression vector. The resulting construction is 1E10VKhu-PAG4622.

For expression of the humanized antibody 1E10, NS-0 cells, 1E10VHhu-PAH4604 and 1E10VKhu-PAG4622 were infected.

The 1E10hu antibody is infected by electroporation and detection for chimeric antibodies as described above.

Example 6. Biological Activity of Chimeric MAb 1E10

The specific binding to the antigen was measured by ELISA to determine the biological activity of the chimeric Mab 1E10.

For the recombinant Mab 1E10 antibody, the microtiter plates were coated with Mab P3. After washing with PBST (saline phosphate buffered solution containing 0.05% Tween 20), nonspecific binding was blocked with bovine sera albumin (BSA) 1% in PBST and incubated for 1 hour at 37 ° C. The wells were washed with PBST and incubated for 1 hour at 37 ° C with purified recombinant Mab 1E10. The wells were washed with PBST, goat anti-human antibody bound to alkaline phosphatase was added and incubated for 1 hour at 37 ° C. Finally, the wells were washed with PBST and substrate buffer containing rnitrophenylphosphate was added. After half an hour, the absorbance at 405 nm was measured.

Chimeric Mab C5 is used for negative control.

Figure 10 shows the specific binding of chimeric Mab 1E10 to Mab P3.

Description of the attached figures

Figure 1: VHP3 DNA and reduced amino acid sequences. The sequences are arranged according to Kabat numbering (Kabat et al. (1991): Sequences of proteins of immunological interest, Fifth Edition, National Institute of Health). The dotted lines are labeled CDRs.

Figure 2: VKP3 DNA and reduced amino acid sequences. The sequences are arranged according to Kabat numbering (Kabat et al. (1991): Sequences of proteins of immunological interest, Fifth Edition, National Institute of Health). The dotted lines are labeled CDRs.

Figure 3: VHP3 is arranged in the most homologous human sequence. The amphipathic segments are underlined and the CDRs are blackened.

Figure 4: VKP3 is arranged in the most homologous human sequence. The amphipathic segments are underlined and the CDRs are blackened.

Figure 5: Specific binding to GM3 (NeuGc) by Mab P3. Different concentrations of Mab P3 and MAb T1 (negative control) were tested by ELISA. Microtiter plates were coated with GM3 (NeuGc) and GM3 (NeuAc) (negative control) ganglioside in methanol and specific binding was measured.

Figure 6: VH1E10 DNA and reduced amino acid sequences. The sequences are arranged according to Kabat numbering (Kabat et al. (1991): Sequences of proteins of immunological interest, Fifth Edition, National Institute of Health). The dotted lines are labeled CDRs.

Figure 7: VK1E10 DNA and reduced amino acid sequences. The sequences are arranged according to Kabat numbering (Kabat et al. (1991): Sequences of proteins of immunological interest, Fifth Edition, National Institute of Health). The dotted lines are labeled CDRs.

Figure 8: VH1E10 is arranged with the most homologous human sequence. The amphipathic segments are underlined and the CDRs are

blackened.

Figure 9: VK1E10 is arranged with the most homologous human sequence. The amphipathic segments are underlined and the CDRs are

blackened.

Figure 10: Specific binding of murine MAb P3 via chemirin Mab 1E10. Different concentrations of Mab 1E10 and MAB C5 (negative control) were tested by ELISA. The microtiter plates were coated with MAb P3 and MAb A3 (negative control) and specific binding was measured.

Claims (16)

1. A chimeric monoclonal antibody derived from a mouse MAb P3 monoclonal antibody that recognizes gangliosides containing N-glycolated sialic acid, and which is produced by a hybridoma cell line with ECASC entry number 94113026, characterized in that the regions are hypervariable CDRs of its heavy and light chains include the following sequences: HEAVY CHAIN CDR1.RYSVH 0 CDR2: MIWGGGSTDYNSALKS CDR3: SGVREGRAQAWFAY LIGHT CHAIN CDR1: KASQDVSTAVA CDR2: SASYRYT CDR3: QQHYSTPWT Chimeric monoclonal antibody according to claim 1, characterized in that the framework regions (FR) of its heavy and light chains include the following sequences: O HEAVY CHAIN FR1: QVQLKESGPGLVAPSQSLSITCTVSGFSLS FR2: VWRQPPGKGLEWLG FR3: RLSISKDNSKSQVFLKMNSLQTDDTAMYYCAR FR4: WGQGTLV LIGHT CHAIN FR1: DIVMTQSHKFMSTSVGDRVSITC FR2: WYQQKPGQSPKLLIY FR3: GVPDRFTGSGSGTDFTFTISSVQAEDLAVYYC FR4: FGGGTKL Monoclonal antibody according to claims 1 and 2, characterized in that for its humanization and storage of its binding properties to the antigen, it comprises at least one of the following substitutions: LIGHT CHAIN Item 8: His replaced by Pro Item 9: Lys replaced by Ser Item 10: Phe replaced by Ser Item 11: Met replaced by Leu Item 13: Thr replaced by Ala Monoclonal antibody according to claims 1 to 3, characterized in that the heavy chain constant region includes the amino acid sequence of the gamma-1 chain and the light chain constant region includes the amino acid sequence of the kappa chain, both derived from human immunoglobulins . 5. A cell line producing any of the monoclonal antibodies according to claims 1 to 4. A pharmaceutical composition for the treatment of malignant breast tumors and melanomas, their metastases and recurrence, comprising any of the monoclonal antibodies according to claims 1 to 4. 7. A pharmaceutical composition for localizing and identifying malignant tumors of the breast and melanomas, their metastases and renewal, characterized in that it comprises any of the monoclonal antibodies according to claims 1 to 4. Use of a monoclonal antibody according to any one of claims 1 to 4 for the manufacture of a medicament effective for the treatment of malignant breast tumors and melanomas, their metastases and recurrence. 9. A chimeric monoclonal antibody derived from the anti-idiotypic mouse monoclonal antibody 1E10 that recognizes the monoclonal antibody of a mouse MAb P3 produced by a hybridoma cell line with entry number ECACC 97112901, characterized in that it is hypervariable and variable. the following sequences: HEAVY CHAIN CDR1: SYDIN CDR2: W1FPGDGSTKYNEKFKG CDR3: EDYYDNSYYFDY LIGHT CHAIN CDR1: RASQDISNYLN CDR2: YTSRLHSG © CDR3: QQGNTLPWT Monoclonal antibody according to claim 9, characterized in that the framework regions (FR) of its heavy and light chains include the following sequences: HEAVY CHAIN FR1: QVQLQQSGAELVKPGASVKLSCKASGYTFT FR2: WVRQRPEQGLEWIG FR3: KATLTTDKSSSTAYMQLSRLTSEDSAVYFCAR FR4: WGQGTTLTV © LIGHT CHAIN FR1: DIQMTQTTSSLSASLGDRVTISC FR2: WYQQKPDGTVKLUY FR3: VPSRFSGSGSGTDYSLTISNLEQEDIATYFC FR4: FGGGTKLESK Monoclonal antibody according to claims 9 and 10, characterized in that for its humanization and storage of its binding properties to the antigen, it comprises at least one of the following substitutions: LIGHT CHAIN Item 7: Thr replaced by Ser Item 8: Thr replaced by Pro Item 15: Leu replaced by Val HEAVY CHAIN Item 5: Gin replaced by Val Item 40: Arg replaced by Ala Item 42: Glu replaced by Gly Item 87 (83 according to Kabat numbering): Thr replaced by Arg Monoclonal antibody according to claims 9 to 11, characterized in that the heavy chain constant region includes the amino acid sequence of the gamma-1 chain and the light chain constant region includes the amino acid sequence of the kappa chain, both derived from human immunoglobulins . A cell line producing any of the monoclonal antibodies according to claims 9 to 12. A pharmaceutical composition for the treatment of malignant tumors of the breast and melanomas, their metastases and renewal, characterized in that it comprises any of the monoclonal antibodies according to claims 9 to 12. A pharmaceutical composition for the in vivo localization and identification of malignant breast tumors and melanomas, their metastases and renewal, comprising any of the monoclonal antibodies according to claims 9 to 12. Use of a monoclonal antibody according to any one of claims 9 to 12 for the manufacture of a medicament effective for the treatment of malignant breast and melanoma tumors, their metastases and recurrence.